The eukaryotic ATP-ases of the SMC (structural maintenance of chromosomes) family form several essential eukaryotic protein complexes that determine higher-order chromosome structure and dynamics in eukaryotic cells. One of these complexes, condensin, represents the main molecular machinery of chromosome condensation, a process indispensable for proper segregation of sister chromatids during anaphase. At present, the molecular mechanism of condensin activity in vivo is unknown. To understand the essence of condensin activity in chromatin, we focus on the specificity of condensin targeting to the natural chromatin sites in budding yeast. We took three approaches to investigating the role of genetic and epigenetic factors in recognizing specific chromosomal domains by condensin: the genomic distribution and cell cycle control of condensin binding outside the nucleolus, molecular mechanisms determining the specificity of mitotic condensin targeting to nucleolar chromatin, and post-translational modifications in condensin binding regulation.
Whole-genome analysis of condensin binding sites
Wang, Butylin; in collaboration with Basrai, Lichten
In budding yeast and higher eukaryotes, condensin is composed of five essential subunits: Smc2, Smc4, Ycs5/Ycg1, Ycs4, and Brn1. Our previous studies identified nucleolar chromatin (the rDNA genomic locus) as the major binding site for condensin in S. cerevisiae. In addition, based on genetic and cell biology data, we suspected the existence of non-nucleolar binding sites. To elucidate the genomic distribution and cell cycle control of condensin binding, we conducted immunoprecipitation of chromatin-bound Smc2, followed by hybridization to genomic microarrays (ChIP-chip analysis). As a result, we identified several novel sites of condensin binding. We established that condensin is distributed over chromosomal arms with strong binding peaks approximately every 10 kb (see Figure 12.1). Quantitative PCR validation confirmed that condensin occupies sites across the genome and in the specialized chromatin regions: near telomeres, in heterochromatic regions, and in the zones of converging DNA replication. Comparison of condensin binding throughout the cell cycle revealed a cyclical mode of condensin binding at some sites. During mitosis, condensin was enriched at rDNA and subtelomeric and pericentromeric regions, suggesting that the regions are the predominant targets of the essential condensin function, namely, separation of sister chromatids at the metaphase-to-anaphase transition.
Molecular pathways governing condensin targeting to nucleolar organizer in mitosis
Wang, Yong-Gonzales, Mehta, Butylin, Strunnikov
Our genetic studies established that several pathways determine proper condensin localization to specific chromatin domains and thus facilitate/regulate the chromosome condensation process. Screening for molecular mechanisms determining specificity of mitotic condensin targeting to nucleolar chromatin focused on elucidating the role played in condensin affinity by the repeating nature of the rDNA locus. The yeast system presents a unique opportunity to dissect the functional interface between the features of rDNA chromatin and the condensin function, as it is possible to rearrange the tandemly repeated NOR locus so that all rRNA genes are episomal. In addition, it is possible to manipulate the copy number of tandem chromosomal rDNA repeats by means of genetics. Thus, we used two strains with either a deletion of the natural nucleolar organizer (episomal rDNA) or just 20 tandem repeats of the 9-kb rDNA unit. We assayed the cells for condensin binding to rDNA by using quantitative ChIP in the course of cell cycle progression. As a result, we identified four robust condensin binding sites within the rDNA and found that two independent pathways control the sites. One pathway involves the transcriptional proficiency of the rDNA cluster, allowing condensin binding only to the rRNA genes that are not transcribed in the given cell cycle. The pathway is activated in mitosis and thus is a likely regulator of the essential condensin function. The second pathway controls condensin binding to the non-transcribed spacer in the rDNA and is independently involved in the interphase function of condensin, probably maintaining the structural integrity of nucleolar organizer. The molecular components of the two pathways are under investigation.
Strunnikov AV. A case of selfish nucleolar segregation. Cell Cycle 2005;4:113-117.
Analysis of post-translational modifications regulating condensin binding to chromatin
Takahashi, Yong-Gonzales; in collaboration with Kikuchi
It has been established that, in all eukaryotes, post-translational modifications contribute to condensin-chromatin binding regulation and condensin activity. We showed that, in budding yeast, the sumoylation pathway (Smt3 conjugation) regulates condensin targeting to the nucleolus in mitosis. The sumoylation target facilitating this regulatory mechanism remains unknown. Using an in vitro biochemical approach, we demonstrated that condensin is not modified by Smt3 when bound to chromatin while topoisomerase II, which interacts with condensin both physically and functionally, can be efficiently modified by Smt3. Our studies established that Smt3 modifications at the Top2 tail can alter targeting of this enzyme within the cell in a chromatin-domain specific manner depending on the number of Smt3 molecules attached. Moreover, genetic analysis of interaction between Top2 modification and the genes encoding the Smt3 E3 (SIZ1 and SIZ2) demonstrated that Top2 mediates the role of these genes in chromosome stability. We also found genetic interaction between the two genes and condensin mutations. We are currently investigating the molecular interface between condensin and sumoylated Top2 as well as condensin sumoylation in vivo.
Strunnikov AV. SMC complexes in bacterial chromosome condensation and segregation. Plasmid 2005 [Epub ahead of print].
Takahashi Y, Yong-Gonzales V, Kikuchi Y, Strunnikov AV. The SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by their role in sumoylation of topoisomerase II. Genetics 2005 [Epub ahead of print].
Wang BD, Eyre D, Basrai M, Lichten M, Strunnikov A. Condensin binding at distinct and specific chromosomal sites in the Saccharomyces cerevisiae genome. Mol Cell Biol 2005;25:7216-7225.
Wang BD, Yong-Gonzalez V, Strunnikov AV. Cdc14p/FEAR pathway controls segregation of nucleolus in S. cerevisiae by facilitating condensin targeting to rDNA chromatin in anaphase. Cell Cycle 2004;3:960-967.
collaborators
Munira A. Basrai, PhD, Genetics Branch, NCI, Bethesda, MD
Yoshiko Kikuchi, PhD, University of Tokyo, Tokyo, Japan
Michael J. Lichten, PhD, Laboratory of Biochemistry, NCI, Bethesda, MD
For further information, contact strunnik@box-s.nih.gov.